Dialyzed fbs

Amino acid starvation treatment was carried out by incubating cells in HBSS buffer (Lonza) with 10% dialyzed FBS (Sigma Aldrich). Samples were collected at indicated time points.

For SILAC experiments HeLa cells were cultured for 5 days at 37 °C in customized double omission DMEM media (AthenaES) supplemented with [¹³C₆]Arg and [¹³C₁,D₃]Met (Cambridge Isotope Laboratories, Inc.) and 10% dialyzed FBS (Sigma) before release into light DMEM media with 10% dialyzed FBS. Cells were harvested at 0, 4, 8, 24 and 48 h after release and flash frozen in liquid nitrogen and stored at −80 °C before sample preparation. For non-SILAC experiments HeLa cells were cultured in DMEM media and treated with 0.5 mM mimosine, 0.15 mM Desferoxamine, 0.15 mM CoCl₂ or 0.25 mM FeSO4·7H₂O for 24 h prior to harvesting. Histones were recovered from isolated nuclei using 0.4 N sulfuric acid extraction and were chemically derivatized using propionic anhydride and digested with trypsin, as previously described [36 (link)]. Briefly, about 5 μg of total histones were dissolved in 10 μl of 50 mM (NH₄HCO₃^, mixed with 20 μl of reaction mixture (3:1 of methanol : propionic anhydride), ammonia was added to ensure pH >7, usually 5 μl, and the sample was incubated at 50 °C for 20 min. After two rounds of derivatization, histones were digested with 0.5 μg of trypsin at 37 °C for 16 hr. After an additional two rounds of derivatization, the digested peptide was diluted in 0.1% TFA.

A2780 cells were placed in glucose-free DMEM media (HyClone) supplemented with 10 mM [U-¹³C6] d-glucose (Cambridge Isotopes, Tewksbury, MA) and 10% dialyzed FBS (Sigma Aldrich, St. Louis, MO). Cells were treated with 10 µM NCL-240 or 0.5% DMSO for 16 hr. Cells were lysed in 0.5 ml of cold methanol and further mixed by vortexing with 0.5 ml cold chloroform and 0.5 ml of water. Polar metabolite extracts were collected after 15 min of centrifugation at 14,000 rpm at 4 °C. Polar extracts were dried in SpeedVac and resuspended in 50 mM phosphate D₂O buffer pH 7.0 with 0.2 mM of NMR standard (DSS, 4,4-dimethyl-4-silapentane-1-sulfonic acid). ¹H NMR spectra of each sample were collected at 25 °C on a Bruker Avance 600 spectrometer using 128 scans and a NOE1D pulse sequence. ¹³C NMR was collected using 20,000–40,000 scans, a 45° degree flip angle, a delay time of 0.5 sec. and an acquisition time of 0.47 sec. ¹H-¹³C 2D spectra were collected using an HSQC pulse sequence. Compounds were identified by matching 1D and 2D NMR data to the HMDB database (Wishart et al., 2009 (link)). The ¹H NMR data were processed and analyzed using CHENOMX NMR Suite 8.0 to identify the compounds present and to measure their concentrations.

For non-titration related experiments, RPMI-1640 (Corning) was supplemented with 10% FBS (ThermoFisher) and penicillin/streptomycin (Gibco). Tryptophan-free RPMI-1640 was custom made by Corning, by modifying item #10040 to exclude L-Tryptophan. Tryptophan-free RPMI-1640 was supplemented with either 1 mg/mL BSA (HyClone), 10 µg/mL bovine insulin (Sigma), 5 µg/mL iron-saturated transferrin (Sigma) and penicillin/streptomycin, or 5% dialyzed FBS (Gibco) and penicillin/streptomycin. For L-Arginine, L-Leucine and L-Lysine titration experiments, SILAC RPMI-1640 (Sigma) was supplemented with 10% dialyzed FBS and penicillin/streptomycin. Cell culture grade amino acids were purchased from Sigma.